1. Field of Invention
The present invention relates to the reading and the detection of the fluorescence of the reaction of the bonding of molecular beacons. The invention is intended as an inexpensive way to determine the results of the bonding of the molecular beacons with an actual DNA strand. These determinations can be used to determine the occurrence of viruses and other molecular presence.
2. Description of Prior Art
Molecular Beacons
Molecular beacons are oligtide probes that can report the presence of specific nucleic acids in homogeneous solutions. These probes were developed by the Public Health Research Institute of the City of New York, U.S. Pat. No. 5,925,517 by Tyagi, Kramer, and Lizardi. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes, such as in real-time monitoring of polymerase chain reactions in sealed tubes or in detection of RNA within living cells. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid. They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence which can be detected.
In order to detect multiple targets in the same solution, molecular beacons can be made in many different colors utilizing a broad range of fluorophores. DABCYL, a non-fluorescent chromophore, serves as the universal quencher for any fluorophore in molecular beacons. Owing to their stem, the recognition of targets by molecular beacons is so specific that single-nucleotide differences can be readily detected. Because of these properties molecular beacons have been used for detection of RNA within living cells for monitoring the synthesis of specific nucleic acids in sealed reaction vessels for homogenous one-tube assays for genotyping single-nucleotide variations in DNA and for multiplex PCR for the detection of four pathogenic retroviruses.
Detection Methods
Presently there are three methods for the reading of the molecular beacons. Using high pressure liquid chromatograph systems, Spectrofluorometers and thermal cyclers with a capacity to monitor fluorescence in real time. These approaches require expensive equipment and are large and complex. Present approaches limit the ability to do testing except at sophisticated laboratories.
3. References and Prior Art Statements
The inventors have also researched the literature and discuss the following patents:
1. U.S. Pat. No. 5,017,475 by Harte and Mastin describes a detection process based on a specific enzyme. The detection of a chemical reaction. The process uses fluorophore-labelled compound. The detection is based on using fluorescent dyes and inks.
2. U.S. Pat. No. 5,574,790 by Liang, Marinello, Ryan, Silverglate and Wray describes a Fluorescence authentication reader that is used with a computer program to determine the user determined and programable encryption of articles"" authentic identity.
3. U.S. Pat. No. 5,666,417 by Liang, Marinello, Ryan and Wray describes a reader that performs the same functions as described in the U.S. Pat. No. 5,574,790 above in No. 2.
4. U.S. Pat. No. 5,686,300 by Berndt describes a fluorescence detection method and apparatus for detecting biological activities in a fluid specimen, such as blood, urine or sputum, where the specimen and a culture medium are introduced into a sealable container and exposed to conditions enabling a variety of metabolic, physical, and chemical changes to take place in the presence of microorganisms in the sample.
5. U.S. Pat. No. 5,719,061 by Rose-Pehrsson and Collins describes a method and apparatus for detection of hydrazine, monomethylhydrazine, and 1,1-dimethylhydrazine in air or other gas mediam or in an aqueous solution.
6. U.S. Pat. No. 5,723,294 by Glass, Coombs, Malmstrom and Wu describes a method and apparatus for permitting multiple PCR-amplified target nucleic acid sequence hybrids within a single sample, labeled with different fluorescent dyes, to be spectrally distinguished using data directly from a fluorescence reader instrument.
7. U.S. Pat. No. 5,861,256 by Glass, Coombs, Malmstrom and Wu describes a method and apparatus that performs the same function as U.S. Pat. No. 5,723,294 described above in No. 6.
The molecular beacon reader allows the operator to view biological samples which have been treated with molecular beacon fluorophores to determine if the samples contain the material for which the molecular beacon is designed. The device illuminates the samples with a specific wavelength of light to excite the molecular beacons. There is a viewing port for the operator to observe the samples and also to photograph them. The device provides the proper light filters to discriminate between the excitation wavelength and the fluorescence. Provision is made for multiple samples to be read simultaneously.